The present invention relates to an immortalized glucose responsive xcex2-cell line. Such cell lines are particularly useful in the treatment of type 1 diabetes in humans but insulin control in other mammals is envisaged as well as cross species Production and use.
In type 1 (insulin dependant) diabetes mellitus the principal defect is the destruction of pancreatic xcex2-cells with the resultant loss of insulin production. The condition is normally controlled by the administration of insulin. However, presently available procedures for insulin delivery do not reinstate the normal regulation of glucose metabolism. In non-diabetic people the secretion of insulin is characterised by a basal secretion which occurs between meals and during the night, and secretion that is stimulated in response to a meal. Commercial preparation of fast-acting (NRI-HI) and slow-acting (ultralente) insulins fail to mimic these natural patterns of secretion in diabetic patients. The fastest acting insulin preparations have a slower onset and more prolonged effect than stimulated insulin secretion, while slow acting insulins produce levels of insulin elevated far above those seen during basal secretion. The net result is that patients on subcutaneous insulin injections have a great difficulty in controlling their glycaemia without risk of significant hyperglycaemia, hypoglycaemia or hyperinsulinaemia. The results of the Diabetes Control and Complication Trial (DCCT) have shown that stringent glycaemic control can significantly reduce the appearance of long-term complications such as nephropathy, retinopathy, neuropathy and cardiovascular disease. Hypoglycaemia is unacceptable and dangerous, while hyperinsulinaemia is associated with a higher risk of atherosclerosis. This is discussed in a background paper, Clinical Science (1997) 92,321-330.
It has thus been clear for many years that there is a requirement for the development of new approaches to improving glycaemic control in diabetic patients. In combination with improved methods for self-monitoring of blood glucose concentrations, open-loop and closed-loop continuous insulin infusion devices have been developed to provide near-normal glycaemia. However, the use of these devices is compromised because: a) some are inappropriate for ambulatory patients; b) they are not fail-safe; c) they do not provide a lophysiological replacement of normal amounts of insulin; and d) they necessitate a high degree of motivation and extensive training on the part of the patient.
The ideal treatment for type I diabetes would be the replacement of the patient""s destroyed xcex2-cells with transplanted pancreatic Islets of Langerhans. However this approach has two major drawbacks: a) the patients have to be treated with immunosuppressants to prevent rejection of the transplanted islets; and b) it is dependent on the availability of tissue from human cadavers. In addition the ability to prepare pure islets from such donor tissue has proved problematic. Porcine Islets have been suggested as a convenient alternative. However, problems have arisen as a result of the purity of porcine Islet preparations, tissue availability, storage, and transplant rejection.
An alternative approach to treatment, which might improve the control of circulating glucose concentrations, is the implantation of cells which have been genetically modified in vitro to express insulin. Accordingly, cultured cell lines might be manipulated to express insulin by DNA-mediated gene transfer, and the cells encapsulated and implanted into the patient. Several approaches have been adopted:
1) Engineering xcex2-cell lines: a number of rodent or hamster xcex2-cell lines are in use. These have been generated by X-ray irradiation of isolated Islets of Langerhans, transformation of isolated Islets of Langerhans by DNA tumour viruses, expression of the SV40 large T antigen in xcex2-cells of transgenic mice or by cell fusion of Islets of Langerhans with immortalized cell lines. The problem with these cell lines is that they tend to dedifferentiate after a period of time in continuous culture. This results in a loss of glucose sensitive insulin secretion from the physiological range (4-10 mM) to a sub millimolar range. Also, because these cells are highly proliferative it is difficult to predict (based on animal studies) the levels of insulin secreted when implanted into diabetic patients i.e. as the cells proliferate within the animals the insulin levels become too high and the animals become hypoglycaemic.
2) Engineering non-xcex2 neuroendocrine cells. Cells such as the mouse corticotrophic cell line AtT20 can be stably transfected with insulin. However, although these AtT20ins cells efficiently process proinsulin to insulin, they do not secrete insulin in response to glucose. Attempts to make AtT20ins cells glucose responsive by transfecting with the glucose transporter GLUT-2 (to generate AtT20insGLUT-2 cells) have been unsuccessfulxe2x80x94the cells show some response to glucose but in the sub-physiological range. A major problem with the use of neuroendocrine cells is that endogenous neuropeptides or hormones (e.g. ACTH) that are co-secreted with insulin may antagonise the effects of insulin or otherwise upset the metabolic balance of the patient. Thus we have shown that although some degree of glycaemic control can be achieved by implanting AtT20ins or AtT20insGLUT-2 cells in diabetic animals, after a period of time hyperglycaemia occurs because the animals have become insulin resistant as a consequence of the elevated ACTH levels secreted by the implanted cells.
3) Engineering non-neuroendocrine cells. Muscle, liver and fibroblast cells have been used to administer insulin. The problem with these cells is they do not have the capacity to process proinsulin to insulin or to sense changes in circulating glucose levels. The first problem has been overcome by mutating the proinsulin cleavage sites to the recognition sequence for the ubiquitously expressed protease furin. The second problem regarding engineering glucose sensitive insulin secretion in non-neuroendocrine cells may be beyond the scope of present technologies and knowledge. However, a constitutive trickle release of insulin from these cells may have therapeutic value under certain circumstances.
4) The final approach is to engineer primary cells taken from diabetic patients. These cells would be of non-neuroendocrine origin, i.e. muscle or fibroblast. The aim would be to attain a stable trickle release of insulin using the mutant proinsulin molecule engineered for cleavage by furin.
Despite progress in the above areas no-one to date has engineered or cloned a cell line which grows well in culture and which secretes insulin in response to changes in glucose concentration in the physiological range. Such a human pancreatic xcex2-cell line has long been sought. We have now succeeded in generating a number of such cell lines using a novel approach based on engineered xcex2-like cells inter alia isolated from patients with persistent hyperinsulinaemic hypoglycaemia of infancy PHHI (also known as nesidioblastosis, nesidioplasia of the pancreas, persistent neonatal hyperinsulinism, congenital hyperinsulinism, familial hyperinsulinism (with hypoglycaemia), persistent infantile hyperinsulinism, hyperinsulinaemic hypoglycaemia, microadenomatosis, islet cell hyperplasia, focal hyperinulinism, diffuse hyperinsulism, glucokinase upregulation disorder, glyceraldehyde dehydrogenase disorder, syndrome of hyperinsulinism and hyperammonemia, insulinoma of childhood).
Although we have used cells derived from PHHI, it will be appreciated that immature cells from other sources or bio-engineered cell lines may be used so long as they give rise initially to immortalized unregulated insulin secretion. By the term immortalized is meant a cell line which proliferates in vitro in culture. Islet cells isolated from some patents with PHHI are ideal for producing a cell line because they spontaneously proliferate in vitro.
By cell line we include cell lines derived from cell lines of the present invention and into which cell DNA from the patented cell line has been incorporated.
Persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI), or nesidioblastosis, although a rare disorder characterised by unregulated insulin secretion and profound hypoglycaemia in infancy and childhood, is the most common cause of persistent hypoglycaemia in childhood. PHHI arises from developmental and dysfunctional abnormalities of the pancreatic xcex2-cells. Newly born children with the disease can suffer severe brain damage if not diagnosed and treated immediately. Treatment in severe cases usually involves partial or even total pancreatectomy.
There is controversy over the aetiology of the disease. The term nesidioblastosis (PHHI) was coined in recognition of the histological appearance of endocrine cells lying in duct epithelium with an apparent failure to aggregate into discrete Islets of Langerhans. However, severe hyperinsulinaemia can also occur in the presence of apparently normal Islets, and doubt has been expressed over the concept that the condition is entirely due to a defect in xcex2-cell differentiation. The disease occurs either in families, particularly in the Middle East, or sporadically.
We have accordingly made two discoveries which enable us to produce an insulin secreting cell line from such an immature source and subsequently to repair the deficiency in the cell line by genetic engineering to give a glucose responsive human xcex2-cell line. According therefore to a first aspect of the present invention there is provided an immortalized insulin producing human xcex2-cell line. This cell line is then bio-engineered to be glucose responsive. The cell line may also be cloned or otherwise proliferated subsequently.
This cell line is preferably derived from immature Islets of Langerhans from a foetal or child donor and may be conveniently derived from a patient with PHHI. Subsequently such a cell line may be genetically engineered to induce glucose responsiveness within the physiological range of 4 to 10 mM. One way of producing this is to transfect the insulin producing cloned human xcex2-cell line with the homeodomain factor PDXI (also referred to as IUF1) and subsequently stably co-transfecting the resultant product with cDNA encoding SUR1 and optionally Kir 6.2 (components of the KATP channel). The resultant products may then be rendered into a form which is implantable e.g. may be encapsulated, or used for screening of novel drug targets for PHHI and other glucose regulation disorders. Specifically the cell line of the invention may be used for at least one of the following:
(a) screening of cationic or anionic selective ion channels;
(b) screening of intracellular and/or cytosolic concentration of calcium ions;
(c) screening of processes for exocytosis or endocytosis of secretary granules;
(d) screening of processes for cell division and/or differentiation;
(e) screening of processes for glucose-induced insulin release via depolarization-response coupling events, or augmentation pathways;
(f) screening of processes for glucose-induced insulin gene transcription or gene transcription associated with diabetes PHHI or insulinomas;
(g) screening of processes for elucidation of xcex2-cell specific ion channels or receptors therefor; and
(h) screening of processes for elucidation of the pharmacology of ion channel modulation proteins.
In a further aspect of the invention there is provided a method for the production of an immortalized glucose responsive insulin producing human xcex2-cell line which comprises selecting an unregulated immortalized human insulin secreting xcex2-cell line, transfecting said selected cell line with elements for the genetic control of glucose responsiveness and proliferating the transfected cell line. Such cells may be subjected to the further steps of genetically engineering the resultant cell line to be glucose, responsive within the physiological range by transfecting with a cDNA encoding PDX1 and subsequently stably co-transfecting the resultant product with cDNA encoding both SUR 1 and Kir 6.2 to form an NISK 9 cell line.
The NISK 9 cell line so produced has been deposited under the provisions of the Budapest Treaty under No. 9709106 at The European Collection of Cell Cultures; Centre for Applied Microbiology and Research, Salisbury, Wiltshire, United Kingdom on Sep. 1, 1997.
The NES2Y cell line is deposited under No. 98081006 at the European Collection of Cell Cultures; Centre for Applied Microbiology and Research, Salisbury, Wiltshire, United Kingdom on Sep. 14, 1998.
According to other embodiments of the present invention there are provided methods of treatment of the human body with PHHI, methods for the treatment of the human body with diabetes, and a method for the treatment of the human body with a condition resulting from abnormal insulin secretion. In this respect, the method of treatment may comprise producing in vitro an immortalized insulin producing human xcex2-cell line which is glucose responsive and thereafter implanting the cells from the cell line into the human body.
Conveniently the cell line is derived from immature, that is to say foetal or a younger child, Islets of Langerhans. Preferably the immature Islets of Langerhans are from the body to which treatment is to be effected. If the immature Islets of Langerhans are taken from the same body as they are implanted into there is a reduced likelihood that they will be rejected.
In a preferred embodiment the Islets of Langerhans are from a foetus or a child with PHHI. Such Islets of Langerhans produce a cell line which is immortal and stable.
Alternatively, the cell line may be derived from a cell-line bio-engineered to produce unregulated insulin secretions.
Preferably the cell line is genetically engineered to be glucose responsive within the physiological range of 4 to 10 mM. One method of producing such a responsive cell line is to transfect the cells with cDNA PXD1 and then to co-transfect cDNA encoding SUR1 and optionally Kir 6.2.